Synthesis of pyrimidines from dinitrogen and carbon

Abstract The element nitrogen and nitrogenous compounds are vital to life. The synthesis of nitrogen-containing compounds using dinitrogen as the nitrogen source, not through ammonia, is of great interest and great value but remains a grand challenge. Herein, we describe a strategy to realize this transformation by combining the heterogeneous approach with the homogeneous methodology. The N2 molecule was first fixed with carbon and LiH through a one-pot heterogeneous process, forming Li2CN2 as an ‘activated’ nitrogen source with high efficiency. Then subsequent homogeneous treatments of Li2CN2 to construct the organic synthon carbodiimide and the RNA/DNA building block pyrimidines were fulfilled. By using 15N2 as the feedstock, their corresponding 15N-labeled carbodiimide and pyrimidines were readily obtained. This homogeneous–heterogeneous synergy strategy will open a new chapter for N2 transformation.


INTRODUCTION
The activation and transformation of dinitrogen (N 2 ) gas are among the most intriguing challenges of modern chemistry. Chemists have explored this field with great effort from different perspectives. Still, until today, the Haber-Bosch process developed at the beginning of the twentieth century remains the dominant way to provide 'activated' nitrogen sources for human society (Fig. 1a) [1,2]. Nowadays, almost all synthetic nitrogenous compounds are synthesized from NH 3 . In other words, virtually all the N atoms in artificial N-containing compounds in human society are from the nitrogen in NH 3 . However, the current Haber-Bosch process and the applied routes from NH 3 to various desired N-containing products are still far from satisfactory in many aspects [3][4][5][6].
An attractive alternative is to construct C-N bonds directly from N 2 and carbon without using NH 3 as the intermediate (Fig. 1b) [7][8][9][10][11][12]. Although this route can be considered an ideal direct use of N 2 besides the Haber-Bosch process and several notable examples have been reported [13][14][15][16][17][18][19][20][21][22], this field is still in its infancy. The biggest problem holding back the field is finding out effective strate-gies that can ingeniously combine the activation of dinitrogen, a notoriously stable molecule, with the formation of nitrogen-carbon bonds.
Looking back through the literature, the most potent means of dinitrogen fixation are based on heterogeneous chemical reactions. In the meantime, the most effective ways to construct various C-N bonds are the homogeneous synthetic methodology. Thus, we envisage that combining a heterogeneous approach with a homogeneous synthetic methodology may be an efficient strategy to achieve the above goal (Fig. 1b). That is, as a concept (Fig. 1c), the N 2 molecule is first turned into an active-enough nitrogenous species (N * ) through a heterogeneous process and then a subsequent homogeneous reaction is performed to construct complex nitrogencontaining organic compounds, taking advantage of both research methodologies. As a proof of concept (Fig. 1d), in this work, the N 2 gas was first fixed with carbon (expanded graphite) and LiH through a heterogeneous process, forming the vital reactive intermediate Li 2 3 . Almost all synthetic nitrogenous organic compounds are synthesized from NH 3 . (b) Synthesis of N-containing organic compounds directly from N 2 molecule and carbon sources. (c) Our general strategy to achieve the above goal: taking advantage of the heterogeneous process to generate active intermediate nitrogenous species, followed by taking advantage of the homogeneous synthetic methodology to make value-added organic compounds. (d) This work is a proof of concept. Through a heterogeneous process, the N 2 gas was first fixed with carbon and LiH to form reactive Li 2 CN 2 , which was then transformed into nitrogen-containing organic compounds through the homogeneous synthetic methodology.
nitrogen-containing organic compounds, including the organic synthon carbodiimide and pyrimidines. As the immediate building blocks for the synthesis of RNA/DNA, pyrimidines are indispensable raw materials in biomedical research, as well as essential elements in the preparation of biological drugs [23][24][25]. This cross-disciplinary strategy greatly simplifies the synthetic steps and reduces the production cost. Also, notably, by using the 15 N 2 gas as the feedstock, their corresponding 15 N-labeled carbodiimide and pyrimidines could be readily obtained.

Synthesis of Li 2 CN 2 from LiH, graphite and N 2 gas
It has been reported that hydrides (H − ) play a peculiar role in dinitrogen fixation and hydrogenation to ammonia, where the reactive hydridic hydrogen functions as electron and/or hydrogen carriers [26,27]. Our previous investigations show that lithium hydride (LiH) can cleave NN triple bond, forming lithium imide (Li 2 NH). Subsequently, the imide can be hydrogenated to produce NH 3 [28]. It is interesting to figure out whether the redox chemistry of the Li-N-H system can be extended to N 2 functionalization beyond ammonia, such as the formation of N-C bonds. As a trial, we initially tested the reaction of Li 2 NH and an easily obtained and straightforward inorganic carbon source, i.e. graphite, at elevated temperatures. It was clearly seen that lithium cyanamide Li 2 CN 2 composed of [NCN] 2− and Li + ions could be obtained ( Supplementary Fig. S1), demonstrating the N-C bond formation. This observation inspired us to explore a direct N-C bond formation from N 2 and graphite mediated by LiH via a one-pot route. Thus, we treated a mixture of LiH and C under a 20-bar N 2 atmosphere in a homemade reactor at 550 o C for 5 h (Fig. 2b). Only H 2 was detected as the gaseous product. The solid residue contained Li 2 CN 2 as the major product, as demonstrated by the X-ray diffraction (XRD) and DRIFT characterizations (red lines of Fig. 2c and d). Bench-scale Li 2 CN 2 (in grams) was obtained facilely with a yield of >85%. Further amplifying the production by using a larger reactor equipped with an H 2 /N 2 separation unit would be straightforward. It should be noted that the known methods for Li 2 CN 2 synthesis typically employ reactive nitrogenous compounds (such as NH 3 , Li 3 N, etc.) and carbon-containing compounds (such as melamine, Li 2 C 2 , CO 2 , etc.) as the N and C sources, and are typically accompanied by the generation of solid byproducts (Fig. 2a) [29][30][31][32][33]. The method developed here has clear advantages in simplicity and atom economy. A step further by feeding the mixture of LiH and C with 15 N 2 under 20 bar could also produce 15 N-labeled Li 2 CN 2 with high purity ( Supplementary Fig. S2), providing a facile and costeffective new route for 15 N-labeled C-N-containing raw materials for further organic synthesis.
Albeit that Li 2 CN 2 formation can be interpreted as an overall reaction of 2LiH + C + N 2 → Li 2 CN 2 + H 2 , the course of this gas-solid reaction contains chemistry that is far from well understood. Our temperatureprogrammed reaction coupled with quasi in situ spectroscopy characterization reveals that the H 2 signal appeared at temperatures of >350 o C showing the occurrence of a redox reaction between H(Li), N 2 and C ( Supplementary Fig. S3). However, only at temperatures of >400 o C could the Li 2 CN 2 phase and its characteristic C=N vibration (2035 cm −1 ) be observed by XRD and diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) (Fig. 2c and d) [34]. Meanwhile, diffraction peaks at ∼31 o and ∼52 o , as well as N-H vibration at 3170 cm −1 assignable to a kind of solid solution of lithium imide (Li 2 NH) and lithium nitride-hydride (Li 4 NH) (denoted as Li 2+x NH), appeared [35].
With an increase in temperature, the signals of Li 2+x NH solid solution fall after rising. We suppose that the hydric H of LiH provides electrons to reduce N 2 first and converts itself to H 2 , and then the formed solid solution Li 2+x NH plays a critical role in establishing C-N bonding, i.e. the hydridic H in the solid solution is an electron source for continued N 2 reduction and perhaps also for the activation of C for subsequent C-N coupling. The involvement of hydride species in carbon reduction has also been identified in previous reports [36].

Synthesis of carbodiimide using Li 2 CN 2 as an organic synthon
Lithium cyanamide, Li 2 CN 2 , containing the centrosymmetric [NCN] 2− subunits in its crystal structure [29,37], is generally regarded as an inorganic salt and has been used as the source of [NCN] 2− subunit to synthesize metal cyanamides in solid-state reactions [38][39][40]. However, although the [NCN] 2− subunit can also be considered to have two cumulated C=N bonds and might be used as an organic building block to synthesize complex nitrogen-containing organic compounds, reports in this area are very rare, probably because Li 2 CN 2 is commercially unavailable and organic chemists are unfamiliar with this salt. As our above-described process achieved gramscale (or a much larger amount of) preparation of Li 2 CN 2 effectively, our strategy to combine the heterogeneous approach with the homogeneous synthetic methodology for N 2 activation and transformation was halfway successful. With this grand success in hand, we immediately initiated our demonstrative investigation into the synthetic application of Li 2 CN 2 following the usual homogenous synthetic methodology. Our first attempt was to treat Li 2 CN 2 simply with Me 3 SiCl (TM-SCl), aiming at the synthesis of the corresponding bis(trimethylsilyl)carbodiimide (BTMSC), a useful versatile synthetic intermediate and an important representative carbodiimide derivative. Carbodiimides are a unique class of hetero-cumulene compounds, which are irreplaceable and valuable precursors to synthesize numerous N-heterocycles. Carbodiimide moieties are also present in various drugs and natural products [41]. As illustrated in Scheme 1a, although the BTMSC can be prepared by other methods, such as the dehydration of bis(trimethylsilyl)urea by phosphorus pentoxide [42], the reaction between methylsilyl cyanide and cyanamide [43], or the reaction between dicyandiamide and 1,1,1,3,3,3-hexamethyldisilazane with ammonia sulfate as the catalyst [44], the related reactants in these reactions are complicated. It takes several extra steps to prepare these reagents from more basic raw materials. CaCN 2 can also react with TMSCl to give the BTMSC, but this reaction needs to be carried out in lithium chloride-potassium chloride molten salt at >400 o C and the yield is not high due to the large proportion of impurities in the raw material CaCN 2 [45].
Compared with the reported routes, our twostep synthetic strategy from N 2 and C to obtain the carbodiimides significantly reduces the energy consumption and the number of steps (Scheme 1c). As shown in Supplementary Table S1, we began our investigation by treating the Li 2 CN 2 with TMSCl in dry toluene as the solvent under the N 2 atmosphere at room temperature for 3 h. The corresponding BTMSC was observed in a very low yield (25%, GC yield). Next, the reaction was tested with different solvents such as Et 2 O, THF, acetone, CH 3 CN and hexane at room temperature. Interestingly, the reaction rate was fast when acetone or CH 3 CN was used as the solvent. However, the carbodiimide product will further react with the excessive TMSCl in the system to give a white precipitate, resulting (c) Synthesis of carbodiimide and 15 N-labeled carbodiimide from N 2 and carbon (this work). In the meantime, the 15 N-labeled carbodiimide is a useful NMR probe. For example, it can be used to determine the connectivity and regioregularity of the polycarbodiimides [46]. However, as shown in Scheme 1b, the classic synthetic route of 15 N-labeled carbodiimide from the commercial 15 N source 15 NH 4 Cl is complicated and tedious.
in an overall low yield of BTMSC. Based on these results, we tested the reaction in mixed solvents. When Et 2 O and acetone at 7 : 1 were used as a mixed solvent, the BTMSC could be obtained in high yield (84% GC yield, 80% isolated yield) after 1.5 h at room temperature (Supplementary Table  S2). The yield indicated that the reaction could also be used to determine the purity of Li 2 CN 2 .
We next tested the sensitivity of Li 2 CN 2 to protonic solvents. As shown in Supplementary Table S3, Li 2 CN 2 was partially hydrolysed when it was soaked in water or alcohol. However, Li 2 CN 2 was not that sensitive to moisture and air. Li 2 CN 2 was easy to handle as the raw material and it did not require additional protection during most reactions.
By using 15 N 2 gas as the starting material, the corresponding 15 N-labeled Li 2 CN 2 can be prepared in good yields. Subsequently, the 15 N-labeled carbodiimide could be prepared in a similar way and obtained in a high isolated yield (72%). This two-step synthetic method has apparent advantages over traditional ways.

Synthesis of pyrimidines using Li 2 CN 2 as an organic synthon
With the above successful synthetic application of Li 2 CN 2 , we tried to challenge a more complicated transformation of Li 2 CN 2 to obtain more valueadded nitrogen-containing organic compounds such as pyrimidines, since pyrimidines are not only immediate building blocks for RNA/DNA but also indispensable raw materials in biomedical research and biological drug discovery [23][24][25]. Cytosine and thymine are two of the pyrimidine bases, which can be prepared based on well-established methods from urea [47][48][49]. After carefully optimizing the reaction conditions, we found that Li 2 CN 2 could be hydrolysed into urea in situ using HCl aqueous solution within several hours at 50 o C in high yields (see Supplementary Tables S4 and S5 for more details). It should be noted that urea is unstable to acids and heat. Thus, the heating time and temperature should be controlled for the hydrolysis process. After hydrolysis, water in the reaction bottle was completely evaporated and pyrimidines were then synthesized in the same pot without isolation and purification of urea. As shown in Scheme 2a, following slightly modified known procedures, cytosine was afforded by the condensation of in situ generated urea with 3,3-diethoxypropanenitrile and sodium methoxide in toluene, while thymine was prepared by the acidcatalysed condensation of α-formylpropionate with in situ generated urea in methanol, followed by an intramolecular cyclization under basic conditions. The 15 N-labeled pyrimidines are valuable tools for studying the structural and dynamic features of biomacromolecules. However, the reported ways to synthesize 15 N-labeled pyrimidines are cumbersome and costly, mainly due to the preparation of 15 N-urea from the 15 NH 4 Cl (Scheme 2b) [50]. To prove our strategy, we further expanded the experiments to incorporate 15 N atoms into the pyrimidines from the 15 N-labeled Li 2 CN 2 . After hydrolysis of the 15 N-labeled Li 2 CN 2 with HCl aqueous solution, the 15 N-labeled urea could be obtained in a high isolated yield (74%) simply. Without isolation of the 15 N-labeled urea, the 15 N-labeled pyrimidines could be obtained successfully following the same procedure as mentioned above (Scheme 2c). These results also confirmed that urea was the intermediate in the reaction and the two N atoms in the pyrimidines were originally from the N 2 gas.

CONCLUSIONS
Our homogeneous-heterogeneous synergy strategy for the formation of C-N bonds from N 2 molecules outlined here differs from those previously described strategies. Typically, forming the C-N bonds from N 2 requires the reduction of N 2 to make it nucleophilic enough to react with carbon electrophiles. However, under most conditions, potent reducing agents used in the activation of N 2 are incompatible with the carbon electrophiles. This incompatibility problem may be solved if the element carbon is used directly as the carbon source instead of using a carbon electrophile. In the first step of our strategy, the key synthetic intermediate Li 2 CN 2 was obtained with high selectivity by simply reacting carbon, N 2 gas and LiH. By taking advantage of the heterogeneous approach, the C-N bond was constructed successfully. Whereas Li 2 CN 2 was primarily reported by inorganic chemists several decades ago, it has been regarded as an inorganic metal salt rather than an organic synthon since then. In the second step of our strategy, we demonstrate that Li 2 CN 2 can be transformed into complex N-containing organic compounds. The valuable organic precursor bis(trimethylsilyl)carbodiimide was constructed readily from Li 2 CN 2 in one step. Moreover, cytosine and thymine could also be constructed facilely via Li 2 CN 2 . By using 15 N 2 as the feedstock, the corresponding 15 N-labeled carbodiimide and pyrimidines were also efficiently prepared, which might have a wide range of applications in biochemistry in the future. We believe that our homogeneous-heterogeneous synergy strategy can be used to construct more diversified N-containing organic compounds in the near future.

Preparation of Li 2 CN 2
The Li 2 CN 2 sample was prepared by the calcination of ball-milled mixtures of LiH and expanded graphite under 20 bar of N 2 at 550 o C for 5 h. Feeding the mixture of LiH and expanded graphite with 15 N 2 under similar conditions could produce 15 N-labeled Li 2 CN 2 with high purity.

Materials characterization
XRD patterns were recorded on a PANalytical X'pert diffractometer using a homemade sample cell covered with KAPTON film to avoid air contamination. Temperature-programmed reaction experiments were performed in a quartz-lined stainless-steel reactor and the exhaust gases were analysed using an online mass spectrometer (Hiden HPR20). Fourier transform infrared measurements were conducted on a Brucker Tensor II unit in DRIFT mode with a scan resolution of 4 cm −1 and an accumulation of 32 scans each time.

General information for the organic synthesis
Li 2 CN 2 is stored in a dry and nitrogen-filled glovebox. Tetrahydrofuran (THF) was distilled from sodium-benzophenone in a continuous still under an atmosphere of argon. Toluene, hexane and Et 2 O were purified using an Mbraun SPS-800 solvent purification system. Acetone and acetonitrile were dried over freshly activated molecular sieves (4Å). Other commercially available reagents were used as received without further purification unless otherwise stated. According to the literature procedure, cytosine [47] and thymine [49] were prepared with slight modifications. The synthesis of 15 N-cytosine and 15 N-thymine was similar by using Li 2 C 15 N 2 as the starting material.

SUPPLEMENTARY DATA
Supplementary data are available at NSR online.